In the skin model activating mutations in H-ras, K-ras and N-ras are sufficient to produce the clonal benign squamous papilloma expressing the phenotype of the initiated cell. Utilizing H-ras transduced isolated primary keratinocytes, we have demonstrated that oncogenic Ras upregulates EGFR ligands, establishing a positive feedback loop through EGFR, thus increasing proliferation and simultaneously activating src kinases to tyrosine phosphorylate and inactivate PKC delta, thereby inhibiting PKC-dependent keratinocyte terminal differentiation. Contemporaneous H-ras mediated activation of phospholipase C and PKCalpha establishes a second feedback loop through IL-1alpha and its receptor, activating MyD88 and NFkB to establish a unique gene expression profile characteristic of both initiated cells and papillomas. Interruption of any of these interacting pathways by drugs, knockdown or genetic deletion reduces or prevents tumor formation. An important downstream effector of these interacting pathways is the upregulation and release of chemokines such as CXCL1 and CXCL2 that act in a paracrine mode to attract inflammatory cells and stimulate angiogenesis and an autocrine mode to promote migration of initiated cells. Interruption of any of these pathways inhibits tumor formation. We have also discovered a fundamental function for Adam17, a disintegrin required for the release of EGFR ligands from their membrane attachment. Knockdown of Adam17 prevents the activation of EGFR in H-ras transformed keratinocytes. Preliminary studies indicate that H-ras mediates Adam17 activity via as yet undetermined action on iRhom1 and iRhom2 that are required for Adam17 membrane localization and maturation. Tissue array analysis of human skin tumors indicates that MET is abundantly expressed in progressing squamous carcinomas along with its ligand HGF. We induced tumors with DMBA and TPA on transgenic mice that overexpress HGF and noted a marked increase in papillomas and carcinomas relative to control groups. Further analysis revealed that activation of MET in epidermis is sufficient to initiate tumor formation after TPA promotion in the absence of DMBA. In isolated keratinoctyes, activation of MET mimics the H-ras initiated phenotype reproducing the EGFR and other required feedback loops. However, the application of DMBA to mice with activated MET produces more carcinomas with K-ras mutations than application of DMBA to control mice where H-ras mutations dominate, suggesting that tissue context influences the selection of ras mutations in tumors. RNA profiling of initiated mouse keratinocytes produced by MET or H-ras activation has revealed signature gene expression changes common to both and not previously associated with tumor initiation. Current studies are extending the analysis to K-ras initiated keratinocytes and using knockdown or overexpression approaches to test for critical elements in the common expression profiles essential for initiation and tumor formation. The pathways that have been defined for transforming mouse keratinocytes are also being tested in human keratinocytes transformed by the oncogenic H-, K- and N- ras alleles. Keratinocytes and other epithelial cells may respond to oncogenic stimuli such as mutant ras alleles by activating a senescence program. Current studies indicate that this program in keratinocytes is mediated by the Rho pathway and depends on the activation of p19/Arf and p21. Antagonism of this pathway provides an opportunity to evaluate the cellular biochemistry associated with premalignant progression and malignant conversion.